JP2589693B2 - Method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid - Google Patents

Abstract

A new process of producing L-2-amino-4-(hydroxymethylphosphinyl)-butyric acid (L-AMPB) is now provided, in which 4-(hydroxymethylphosphinyl)-2-oxo-butyric acid (OMPB) is treated with one or more transaminases or with one or more microorganisms capable of producing the transaminase, in the presence of one or more amino-donor compounds such as alpha -amino acids. According to this process, the optically active L-AMPB useful as herbicidal agent can be produced efficiently in a facile way for a reasonable reaction time.

Description

The present invention relates to L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid, which has herbicidal activity and is useful as a herbicide (Japanese Patent Publication No. No. 61-56210 or U.S. Pat.
No. 65,654).

(Conventional technology) As a method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid (hereinafter referred to as L-AMPB) represented by the following formula, L-AMPB is contained as a component in the molecule, and herbicidal activity is determined. SF-1293 substance (also known as Bialaphos)
(See JP-B-51-639 and U.S. Pat. No. 4,309,208), that is, L-2-amino-4- (hydroxymethylphosphinyl) -butyryl-L-alanyl-L-.
There is a method of decomposing alanine by acid (see JP-A-48-85538) or a method of decomposing SP-1293 substance with a microbial enzyme (see JP-A-49-31890). other than this,
Chemical synthesis methods (JP-A-48-91019 and JP-A-54-845)
For example, a method of obtaining AMPB in a racemic form by optical resolution with a microbial enzyme to obtain L-AMPB has been known. Furthermore, in recent years, the present inventors have reported a method of culturing L-AMPB-producing bacteria belonging to the genus Streptomyces and directly collecting L-AMPB from the culture solution (see JP-A-57-47485). ).

In order for a substance having herbicidal activity, such as L-AMPB, to be developed and manufactured as a herbicide, and to be put into practical use as a commercial product, a low-priced product should be developed in accordance with the development research on enhancing the safety and herbicidal efficacy of the substance. In addition, research for improving a production method suitable for industrialization for mass production is essential. AMPB produced by the above chemical synthesis method is composed of L-AMPB and D-AMPB.
Although it is a mixture with AMPB, D-AMPB itself has almost no herbicidal activity and is a non-natural substance, so when applied to soil, it is easily decomposed by soil bacteria and easily remains in the soil when applied to soil. Therefore, it may cause environmental pollution.
In the case where L-AMPB is to be produced by a synthesis method, racemic AMPB is obtained first and then terminated, so that D-AMPB and L-AMPB are separately separated from this racemic form by optical resolution or the like. Separation is required, which is complicated and the yield of L-AMPB is low. However, L- using microorganisms or enzymes
In the method for producing AMPB, only L-AMPB, which is a natural substance, can be obtained. Natural L-AMPB is an ideal herbicide that does not participate in the herbicidal action and is easily decomposed and metabolized by soil bacteria without remaining in the soil. it is conceivable that.

The present inventors have focused on the above problems, and have an advantage of an AMPB synthesis method that can be produced in a large amount and an advantage of an L-AMPB production method using a microorganism that only L-AMPB can be produced. We are trying to combine them and conducting research to establish an improved production method of L-AMPB that can selectively produce L-AMPB in large quantities.

(Means for Solving the Problems) L-AMPB can be regarded as a kind of derivative of α-amino acid. On the other hand, generally, the normal α-
As for amino acids, it is known that α-amino acids are produced by transamination from the corresponding 2-oxo acids by the action of transaminase. Thus, the present inventors have focused on 2-oxo acid corresponding to L-AMPB and have conducted intensive studies. 4- (hydroxymethylphosphinyl) -2 represented by
Treating oxo-butyric acid (hereinafter abbreviated as OMPB) with a certain transaminase or with a certain microorganism capable of producing transaminase in the presence of glutamic acid or a salt thereof as an amino group donor; Have found the surprising fact that optically active L-AMPB can be produced from non-optically active OMPB in a suitably short reaction time with substantial yield.

Therefore, according to the first present invention, 4- (hydroxymethylphosphinyl) -2 represented by
Treating oxo-butyric acid with a microorganism capable of producing one or more transaminases in the presence of glutamic acid or a salt thereof as an amino group donor, but excluding microorganisms of the genus Escherichia. The following equation And a method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid represented by the formula:

In carrying out the first method of the present invention, OMPB is dissolved in an aqueous liquid reaction medium containing OMPB and glutamic acid or a salt thereof acting as an amino group donor.
And glutamic acid or a salt thereof as the amino group donor are allowed to act on a microorganism capable of producing transaminase.

In the first method of the present invention, generally, OMPB to L
The transamination reaction for conversion into -AMPB is preferably carried out in a reaction medium solution having a pH of 7.5 or more, preferably in a range of pH 8.0 to 9.0. The pH is adjusted by adding sodium hydroxide or a suitable buffer. The reaction conditions are preferably such that the production of transaminase involved in the reaction is in the range of the optimum temperature and the optimum pH of the action of the microorganism. Usually, the reaction is at room temperature to
It is good to carry out at 60 ° C, preferably at 20 to 50 ° C.

OMPB used as the raw material is a known substance, and its production method and physicochemical properties are described in, for example, JP-A-56-92.
No. 897 or U.S. Pat. No. 4,399,287. In the first method of the present invention, at the beginning of the reaction, it is usually appropriate that the initial concentration of the raw material OMPB dissolved in the reaction medium is in the range of 0.1 to 100 mg / ml.

Microorganisms used in the first invention include actinomycetes, bacteria, yeasts, and molds. An example of an actinomycete that can be used in the first invention is Streptomyces albus (St.
reptomyces albus), Streptomyces griseus (Streptomyces griseus), Streptomyces hygroscopicus (Streptomyces hygroscopicus), Streptomyces lividans (Streptomyces lividans),
Streptomyces bilidochromogenes
viridochromogenes), Streptomyces pilosus, Streptoverticillium cinnamoneu (Streptoverticillium cinnamoneu)
m), Streptomyces morokaensis (Streptomy
ces morookaensis), Nocardia Mediterrani (Noca)
rdia maditerranei), Nocardiopsis dassonvillei), Saccharopolyspora hirsuta, Kitasatosporia phosaracinea (Kitasatosporia phosa)
lacinea), Micromonospora Carbonacy (Micromo
nospora carbonacae), Streptosporangium pseudovulgare
And so on.

Examples of the bacteria that can be used in the first invention include Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureu.
s), Pseudomonas aer
uginosa), Pseudomonas c
epacia), Serratia marcesc
ens), Corynebacterium glutamicum (Corynebac
terium glutamicum).

Examples of the yeast used in the first invention include Saccharomyces cerevisiae,
Candida albicans, Cryptococcus neoformans
formans), Debaryomyces
hansenii), Trigonopsis variabilis (Trigonop)
sis variabilis, Hansenula
schenggi).

Examples of molds that can be used in the first invention include Aspergillus flavus, Aspergillus terreus, Mucor spinescens, Aureobasidium pullulans, and Caetmium.・ Globosum (Ghaetomium globosum), Penicillium funiculosu
m), Gliocladium viren
s) and the like.

Each of the above-mentioned actinomycetes, bacteria, molds and yeasts can be a type culture deposited at a known microorganism preservation institution and can be purchased therefrom.

Further, preferred examples of the microorganism used in the first method of the present invention include SF-1293 belonging to the genus Streptomyces.
Streptomyces hygroscopicus SF-1293 known as a substance producing bacterium
(FERM BP-130 or ATCC 21705), JP-B-51-639 or U.S. Pat. No. 3,832,394) or a mutant thereof, Streptomyces hygroscopicus SF.
-1293NP-50 strain (FERM P-7804 or FERM BP-1368)
(Japanese Patent Application Laid-Open No. 61-58589 or European Patent Application Publication No. 01733)
No. 27) or Streptomyces lividans
There are 66 strains (FERM BP-737; see JP-A-59-175889 or EP-A-0196375). Others
Any microorganism that can produce an enzyme having a transaminase activity that converts OMPB to L-AMPB in the presence of glutamic acid or a salt thereof as an amino group donor can be used. In addition, the above Streptomyces
Mycological properties of Hygroscopicus sp-1293 strain
No.-639 or U.S. Pat. No. 3,832,394. The mycological properties of Streptomyces hygroscopicus SF-1293 NP-50 strain (FEPM P-7804) are the same as those of the above-mentioned SF-1293 strain, but lack the biosynthesis ability of SF-1293 substance. Genetic characteristics are different in that
58589 or EP-A-0173327). Additionally, Hygroscopicus lividans 66 (FE
The bacteriological properties of RM BP-737) are described in JP-A-59-175889. Of these fungi, the strains with FERM BP-numbers have been deposited with the Institute of Microbial Industry and Technology under the terms of the Budapest Treaty.

According to the second aspect of the present invention, 4- (hydroxymethylphosphinyl) -2 represented by
Treating oxo-butyric acid with one or more transaminases in the presence of glutamic acid or a salt thereof as amino group donor, characterized by the following formula: And a method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid represented by the formula:

The enzyme used in the second method of the present invention, that is, the transaminase, may be any transaminase having transaminase activity capable of converting OMPB to L-AMPB in the presence of glutamic acid or a salt thereof as an amino group donor. May be.

Suitable examples of transaminases used in the second method of the present invention include commercially available glutamic acid-oxaloacetic acid-
Transaminase (abbreviated as GOT) (enzyme international classification number EC2,6,1,1) or glutamate-pyruvate-transaminase (abbreviated as GPT) (enzyme international classification number EC2,6,1,2), Alternatively, there is an enzyme having a transaminase activity that converts OMPB to L-AMPB in the presence of L-glutamic acid as an amino group donor. These transaminases can be used alone or in combination of two or more. Preferably, a commercially available glutamate-oxaloacetate-transaminase (enzyme international classification number EC2, 6, 1, 1) can be used, or G
A microorganism having OT activity, for example, Streptomyces hygroscopicus SF-1293 strain (see JP-A-57-47485;
FERM BP-130; ATCC21705).
A transaminase having GOT activity can be used.

Suitable examples of glutamic acid or a salt thereof as an amino group donor used in the present invention include L-glutamic acid or an alkali metal salt thereof, and D-glutamic acid can also be used as an amino group donor. Before the start of the reaction, the molar ratio of the amount of glutamic acid as the amino group donor to the amount of OMPB in the reaction medium is generally preferably in the range of 10: 1 to 1:10.

In the first and second methods of the present invention, glutamic acid as an amino group donor may be a commercially available product, and may generally be a mixture of D- and L-forms, but is composed of only the L-form. Preferably it is. As a salt of glutamic acid, an alkali metal salt, in particular, a sodium salt or a potassium salt can be used.

In the first method of the present invention, the microorganism is treated with an OM of the formula (II)
When acting on PB and an amino group donor, it is performed in the following manner. That is, first, the microorganism is cultured in a medium containing nutrients used for ordinary microorganism culture. As the nutrient source, a known nutrient used for culturing a normal microorganism is used. For example, as a carbon source,
Glucose, starch, glycerin, shoe cloth, syrup,
Molasses and the like. These may be used alone or in combination. As the nitrogen source, soybean flour, wheat germ, meat extract, peptone, dried yeast, corn steep liquor, ammonium sulfate and the like are used alone or in combination. In addition, if necessary, inorganic salts such as calcium carbonate, salt, potassium chloride, and phosphate can be added. The most suitable culture method is a liquid culture method, particularly a submerged culture method. The cultivation is performed under aerobic conditions, and a suitable temperature for culturing is 25 to 40 ° C. The appropriate number of days for culture is 1 to 4 days for actinomycetes, 1 to 2 days for bacteria, 1 to 2 days for yeast, and 1 to 4 days for mold.

Using the culture solution of the microorganism itself, or, if desired, separating the cells from the culture solution and washing the viable cells, and suspending the cells at a certain concentration in water or physiological saline or an appropriate buffer. Body suspensions can also be used. OMPB (which is the first substrate) is added simultaneously or sequentially with the amino group donor (which is the second substrate) to a culture solution containing the cells of the microorganism to be used or other cell suspensions. Thereafter, the liquid is kept so that the microorganism acts on the OMPB and the amino group donor to advance the OMPB conversion reaction. As described above, the concentration of the bacterial cells in the aqueous reaction medium liquid for converting OMPB and the amino group donor into L-AMPB by treating the microorganism with the microorganism, the concentration of the OMPB and the amino group donor, and the reaction temperature and pH conditions are determined from OMPB. The conversion is appropriately adjusted within a range in which the conversion reaction to L-AMPB proceeds efficiently and the microorganism maintains the state of exerting the function of the microorganism. The reaction time is also adjusted so that a substantial amount of L-AMPB is produced and accumulated in the reaction solution.

In the second method of the present invention, when OMPB and glutamic acid or a salt thereof as an amino group donor are treated with a transaminase, an OMPB and an amino group are added to an appropriate transaminase aqueous solution or an enzyme solution dissolved in a buffer. The OMPB enzyme reaction is performed by adding and dissolving the donor. The respective concentrations of the enzyme, OMPB and amino group donor in the reaction system, as well as the reaction temperature and pH conditions are appropriately adjusted within an appropriate range in which the conversion reaction from OMPB to L-AMPB proceeds efficiently.

As the above-mentioned transaminase, those in the form of a commercially available enzyme product may be used, or the cells may be crushed in a culture solution of actinomycetes, bacteria, molds or yeasts which can be used in the present invention. An aqueous solution of a crude enzyme directly produced by the above method or an aqueous solution obtained by dissolving a crude enzyme in water can be used. Further, a crude enzyme solution in the form of a disrupted cell solution may be used.

Further, even if the crude enzyme solution is extracted from the microorganism or a culture solution thereof by a known method such as ultrasonic treatment or lysozyme treatment, the crude enzyme solution is OM in the presence of an amino group donor.
The ability to produce L-AMPB from PB can be used in the second method of the present invention. In addition, it is natural that an aqueous solution of the purified enzyme can be used. It is known that by immobilizing enzymes and microorganisms with an organic solvent, a cross-linking agent, a carrier, etc., the stability and operability of the enzyme are improved. Immobilized microorganisms can also be used in the method of the present invention if they have the ability to produce L-AMPB from OMPB.

For example, in the second method of the present invention, the enzymatic reaction for converting OMPB to L-AMPB using a transaminase is preferably performed at pH 7.5 or higher, preferably in the range of pH 8.0 to 9.0. The reaction conditions are preferably in the range of the optimal temperature and the optimal pH of the enzyme involved in the reaction.

The collection and purification of L-AMPB from the reaction solution containing L-AMPB produced by the first or second method of the present invention can be carried out by using L-AMPB from a bacterial culture of a conventional L-AMPB fermentation production method. Can be carried out in the same manner as the method for collecting and purifying the same. For details, refer to the description of JP-A-57-47485. That is, L-AMPB was adsorbed on a cation exchange resin such as Dowex 50W (manufactured by Rohm and Haas),
A method of developing and eluting with water or dilute ammonium water can be used. The herbicidal effect of L-AMPB obtained by the method of the present invention was tested, and it was found that the herbicidal effect was equivalent to that of L-AMPB obtained by the fermentation method of JP-A-57-47485. Was done.

Hereinafter, the present invention will be described in more detail by examples,
The present invention is not limited to this.

Example 1 (1) Streptomyces hygroscopicus (Stre
ptomyces hygroscopicus) SF-1293 NP-50 strain (FERM P
-7804 or FERM BP-1368) in preculture medium (soluble starch)
2.0%, 1.0% polypeptone, 0.3% meat extract, 0.05% dipotassium hydrogen phosphate; pH 7.0). This is 28
A culture medium obtained by shaking culture at 24 ° C. for 24 hours was used as a seed mother, and this was used as a seed in a 2% inoculation amount in a production medium (glucose 7.0%, bactosoytone 4.4%, monopotassium phosphate 0.327%, disodium phosphate 0.0852%, dootite TES 1. 15%, cobalt chloride
(0.0001%; pH 6.0), and cultivated at 28 ° C. with aeration and stirring.

(2) 20 ml of a culture solution of Streptomyces hygroscopicus SF-1293 NP-50 strain cultured for 3 days in the production medium as described above, and an OMPB aqueous solution (OMPB concentration 87 mg / ml, adjusted to pH 7.0 in advance) ), 40 ml of a commercially available aqueous solution of sodium L-glutamate (concentration of sodium L-glutamate 170 mg / ml), and 10 ml of 1 M Tris-HCl buffer (pH 8.5) were combined in a 250 ml Erlenmeyer flask. (In this case, the OMPB concentration in the combined solution is about 26 mg / m
l) The reaction was carried out for 24 hours while weakly shaking the mixture in the flask at 37 ° C. Thereafter, the obtained reaction mixture was centrifuged to remove bacterial cells, and the resulting supernatant solution (100 m
l) was analyzed using an amino acid analyzer, and L in the supernatant solution was analyzed.
When -AMPB was detected, it was confirmed that 14 mg / ml L-AMPB was produced.

(3) The supernatant solution (100 ml) obtained when sodium L-glutamate was used as the amino group donor in (2) above was used as a 400 ml Dowex 50 W × 2 (H ⁺ type) cation exchange resin tower. After that, the mixture was developed with dilute aqueous ammonia, and fractions containing L-AMPB were collected and concentrated. Then, this was applied to a 150 ml Dowex 1 × 2 (CH ₃ COO ⁻ type) anion exchange resin tower, washed with water, and eluted with a 0.3N aqueous acetic acid solution.

The fraction containing L-AMPB as the eluent was concentrated to dryness under reduced pressure, and further dried under vacuum to obtain 720 mg of a white powder of L-AMPB. This white powder was crystallized from methanol. The obtained crystal was analyzed by elemental analysis, specific light intensity, melting point, infrared absorption spectrum, nuclear magnetic resonance spectrum, and mass spectrometry by a conventional method, and it was confirmed that the crystal completely matched the L-AMPB sample. Was.

Example 2 (1) Streptomyces hygroscopicus SF-1293 NP-50 strain (FER) cultured in the production medium in Example 1 (1)
20 ml of the culture solution of MP-7804 or FERM BP-1368) was placed in a 30 ml centrifuge tube, and centrifuged at 3,000 rpm for 10 minutes. The precipitated cells were washed with 30 ml of 50 mM Tris-HCl buffer (pH 8.
5) Suspended. 20 ml of this cell suspension, 30 ml of an OMPB aqueous solution (OMPB concentration was 87 mg / ml, which had been adjusted to pH 7.0 in advance), and a commercially available aqueous L-glutamate aqueous solution (sodium L-glutamate concentration 170 mg / ml) ) 40ml
And 10 ml of 1 M Tris-HCl buffer (pH 8.5) for 250
Merging was performed in a ml Erlenmeyer flask (in this case, the OMPB concentration in the merged solution was about 26 mg / ml). This was reacted at 37 ° C. for 24 hours with gentle shaking. Thereafter, the obtained reaction mixture was centrifuged to remove bacterial cells, and the obtained supernatant solution (100 ml) was analyzed using an amino acid analyzer, and L-AMPB in the reaction solution was detected. / ml L-AMPB
Was produced.

(2) The supernatant solution (100 ml) obtained in (1) above was mixed with 400 ml of Dowex 50W × 2 (H ⁺ ) in the same manner as in Example 1 (3).
Mold) and developed with dilute aqueous ammonia. Thereafter, when treated in the same manner as described in Example 1 (3), 750 mg of L-AMPB was obtained as a white powder.

Example 3 (1) Streptomyces hygroscopicus SF-1293 NP-50 strain (FER) cultured in the production medium in Example 1 (1)
20 ml of the culture solution of MBP-1368) was placed in a 30 ml centrifuge tube,
Spin and spin for 10 minutes. 30 ml of the precipitated cells
It was suspended in mM Tris-HCl buffer (pH 8.5). The cell suspension was crushed for 10 minutes using an ultrasonic crusher, and then centrifuged at 10,000 rpm for 10 minutes to obtain 20 ml of a crude enzyme solution as a supernatant.

(2) 20 ml of this supernatant (crude enzyme solution), 30 ml of an OMPB aqueous solution (OMPB concentration was 87 mg / ml, which had been adjusted to pH 7.0 in advance), and an aqueous solution of sodium L-glutamate (L
40 ml of sodium glutamate concentration 170 mg / ml) and 1 M
Combine 10 ml of Tris-HCl buffer (pH 8.5) in a 250 ml Erlenmeyer flask. In this case, the combined liquid
The OMPB concentration will be about 26 mg / ml. This was reacted at 37 ° C. for 24 hours with gentle shaking. Thereafter, the reaction solution (100 ml) was analyzed using an amino acid analyzer, and L-AMPB in the reaction solution was detected. As a result, it was confirmed that 16 mg / ml L-AMPB was produced.

(3) In the same manner as in Example 1 (3), 400 ml of Dowex 50W × 2 (H
⁽⁺ ) Ion-exchange resin tower and developed with diluted ammonia water. Thereafter, when treated in the same manner as described in Example 1 (3), 740 mg of L-AMPB was obtained as a white powder.

Example 4 (1) Streptomyces lividans 66 strain (FERM BP)
-737) was inoculated into 10 ml of a preculture medium having the same composition as that used in Example 1 (1). This was cultured with shaking at 28 ° C. for 24 hours, and used as a seed mother. This was inoculated into a production medium having the same composition as that used in Example 1 (1) at an inoculum of 2%. Thereafter, aeration and agitation culture was performed at 28 ° C.

(2) 20 ml of a culture solution of Streptomyces lividans 66 strain cultured for 3 days in a production medium as described above, and 30 ml of an OMPB aqueous solution (OMPB concentration was 87 mg / ml, which had been adjusted to pH 7.0 in advance). In a 250 ml Erlenmeyer flask, 40 ml of a commercially available aqueous solution of sodium L-glutamate (concentration: 170 mg / ml) and 10 ml of 1 M Tris-HCl buffer (pH 8.5) were combined (OMPB in the combined solution). Concentration is about 26mg / ml
become). This was reacted at 37 ° C. for 24 hours with gentle shaking. Thereafter, the obtained reaction mixture was centrifuged to remove bacterial cells, and the obtained supernatant solution (100 ml) was analyzed using an amino acid analyzer to detect L-AMPB in the supernatant solution. It was confirmed that 6 mg / ml L-AMPB was produced.

Example 5 (1) Streptomyces hygroscopicus SF-12
93 strains (FERM BP-130; ATCC21705) were inoculated into 10 ml of a preculture medium having the same composition as that used in Example 1 (1).
This was cultured with shaking at 28 ° C. for 24 hours, and used as a seed mother. This was inoculated into a production medium having the same composition as that used in Example 1 (1) at an inoculum of 2%. Thereafter, aeration and agitation culture was performed at 28 ° C.

(2) of the culture solution of Streptomyces hygroscopicus SF-1293 strain cultured for 3 days in the production medium as described above;
20 ml, 30 ml of an OMPB aqueous solution (OMPB concentration was 87 mg / ml, which had been adjusted to pH 7.0 in advance), 40 ml of a commercially available sodium L-glutamate aqueous solution (concentration 170 mg / ml), and 1 M
10 ml of Tris-HCl buffer (pH 8.5) was combined in a 250 ml Erlenmeyer flask (OMPB in the combined solution).
The concentration will be about 26 mg / ml). This was reacted at 37 ° C. for 24 hours with gentle shaking. Thereafter, the obtained reaction mixture was centrifuged to remove bacterial cells, and the resulting supernatant solution (100 m
l) was analyzed using an amino acid analyzer, and L in the supernatant solution was analyzed.
When -AMPB was detected, it was confirmed that 7 mg / ml of L-AMPB was produced.

(3) The supernatant solution (100 ml) obtained in the above (2) was mixed with 400 ml of Dowex 50W × 2 (H ⁺ ) in the same manner as in Example 1 (3).
Mold) and developed with dilute aqueous ammonia. Thereafter, when treated in the same manner as described in Example 1 (3), 350 mg of L-AMPB was obtained as a white powder.

Example 6 20 g of a wet cake of commercially available baker's yeast (Saccharomyces cerevisiae) (manufactured by Oriental Yeast) was added to 5 g of a wet cake.
The cells were suspended in 100 ml of 0 mM Tris-HCl buffer (pH 8.5). 20 ml of this baker's yeast cell suspension and an OMPB aqueous solution (OM
PB concentration is 87mg / ml, adjusted to pH 7.0 in advance)
And 30 ml of a commercially available aqueous sodium L-glutamate solution (concentration of sodium L-glutamate 170 mg / ml)
And 10 ml of 1 M Tris-HCl buffer (pH 8.5) for 250
Merging was performed in a ml Erlenmeyer flask (the OMPB concentration in the merged solution was about 26 mg / ml). This was reacted at 37 ° C. for 24 hours with gentle shaking. Thereafter, the obtained reaction mixture was centrifuged to remove bacterial cells, and the obtained supernatant solution (100 ml) was analyzed using an amino acid analyzer, and L-AMPB in the reaction solution was detected. It was confirmed that / ml of L-AMPB was produced.

Example 7 OMPB in 50 mM Tris-HCl buffer (pH 8.5) containing 400 μg / ml OMPB and 600 μg / ml L-glutamic acid
Was reacted with transamylase to produce L-AMPB. As the transaminase, a commercially available glutamic acid-oxaloacetic acid-transaminase (GOT) (Boehringer Mannheim) was used. This GOT concentration is 4
0 units / ml. After performing the enzyme reaction at 37 ° C for 24 hours, 3
The reaction was stopped by heating at 100 ° C for minutes. The resulting reaction solution was adjusted to pH 2 by adding dilute sulfuric acid, and insoluble materials were removed by centrifugation to obtain a supernatant. L-AMPB in supernatant solution
Was quantified by analysis using an amino acid analyzer. The amino acid analyzer was an MTO-type MLC-703 manufactured by Atto Co., Ltd.
Minutes. It was confirmed that the supernatant solution contained 100 μg / ml L-AMPB.

Example 8 (1) Culture method From the seed culture slant, the bacteria shown in the following table were converted into a liquid medium (glucose 0.5%, yeast extract 0.3%,
In a large test tube containing 10 ml of meat extract 1.0%, peptone 1.0%, sodium chloride 0.3%, pH 7.0) (only in the case of cultivation of actinomycetes, a test tube with a stainless steel coil was used) Inoculate the above liquid medium at 28 ° C, 24
Shaking culture was performed for a period of time (or 48 hours).

(2) Preparation of bacterial cells 1 ml of broth of the culture solution (0.2 ml only for mold) was extracted into a micro-test tube (manufactured by Eppendorf), centrifuged at 12000 rpm for 3 minutes, and the supernatant was removed. The liquid was discarded, and the remaining cells were collected as a sample.

(3) Transamination reaction 20 μl of 1 M Tris-HCl buffer (pH 8.0) and O
20μ (pH 8.0) of MPB aqueous solution (OMPB concentration 200mg / ml),
Sodium L-glutamate as an amino group donor;
40 µ of an aqueous solution containing mol / ml and 120 µ of water were added, and allowed to stand at 37 ° C for 24 hours to react. After the reaction, the reaction solution was centrifuged at 12000 rpm for 3 minutes, and then the content of L-AMPB in the supernatant was measured by an amino acid analyzer.

 The results are shown in Tables 1 to 7 below.

Among the microorganisms shown in this specification, particularly in Tables 1 to 7, the ATCC-numbered strains are the American Type Culture Collection (Washington, DC, USA)
The strains that have been deposited with the JCM number and have been assigned a JCM number are RIKEN's Microbial Strain Storage Facility (Japan Collection of Micr
ooganisms (Wako-shi, Saitama) and the IAM-numbered strains are the Institute of Applied Microbiology of the University of Tokyo (Institut
e of Applied Microbiology, University of Tokyo), and the IFO-numbered strains have been deposited at the Institute for Fermentation (Osaka City), and these strains have been deposited in their respective deposits. It is a known type of culture microorganism that can be ordered and obtained from a source.

(Effect of the Invention) According to the present invention, LMP is produced from OMPB by the action of a microorganism having transamylase-producing ability or transamylase in the presence of glutamic acid as an amino group donor or in the presence thereof.
It became clear that if AMPB was produced, L-AMPB could be produced in a high yield, at a low cost, and in a large amount in an appropriate reaction time.

Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12P 13/04 C12R 1:01) (C12P 13/04 C12R 1:07) (C12P 13/04 C12R 1: 265) (C12P 13/04 C12R 1:44) (C12P 13/04 C12R 1:38) (C12P 13/04 C12R 1: 425) (C12P 13/04 C12R 1:15) (C12P 13/04 C12R 1: 85) (C12P 13/04 C12R 1:72) (C12P 13/04 C12R 1:78) (C12P 13/04 C12R 1:66) (C12P 13/04 C12R 1: 785) (C12P 13/04 C12R 1:78) 80) (C12P 13/04 C12R 1: 645) Accession number of microorganisms FERM BP-737 Accession number of microorganisms FERM BP-1368 Accession number of microorganisms FERM BP-130 (72) Inventor Shinji Miyamichi Kohoku-ku, Yokohama-shi, Kanagawa 760 Norooka-cho Meiji Seika Co., Ltd. 760, Meiji Seika Co., Ltd., Pharmaceutical Research Laboratories, Kohoku-ku, Yokohama-shi, Kanagawa Prefecture, Japan (72) Inventor Hiroyuki Anzai 760, Shimooka-cho, Kohoku-ku, Yokohama City, Kanagawa Prefecture Meiji Seika Co., Ltd. 580, Horikawa-cho, Sachi-ku, Kawasaki-shi, Japan Meiji Seika Co., Ltd., Pharmaceutical Development Laboratory (72) Inventor Yachiyo Yoshizawa 580, Horikawa-cho, Sachi-ku, Kawasaki, Kanagawa, Japan Meiji Era Confectionery Co., Ltd. 580, Horikawa-cho, Saiwai-ku, Kawasaki-shi, Kanagawa Prefecture Inside the Pharmaceutical Development Laboratory (72) Inventor Hiroshi Ogawa Meiji confectionery Co., Ltd. 580, Horikawa-cho, Kawasaki-shi, Kanagawa Prefecture (72) Inventor Eiji Takebe Meiji Seika Co., Ltd., Pharmaceutical Development Laboratory (72) Meiji Seika Co., Ltd., Pharmaceutical Development Laboratory (72) Meiji Seika Co., Ltd. Inventor Yukizo Nagaoka Kanagawa 760 Morioka-cho, Kohoku-ku, Hama-shi Meiji Confectionery Co., Ltd. (72) Inventor Shunzo Fukatsu 580 Horikawa-cho, Saiwai-ku, Kawasaki-shi, Kanagawa Prefecture Meiji Confectionery Co., Ltd. 580 Horikawa-cho, Sachi-ku, Kawasaki-shi Meiji Seika Co., Ltd. Drug Development Laboratory (56) References JP-A-62-296886 (JP, A)

Claims (9)

(57) [Claims] 1. The following equation 4- (hydroxymethylphosphinyl) -2 represented by
Treating oxo-butyric acid with a microorganism capable of producing one or more transaminases in the presence of glutamic acid or a salt thereof as an amino group donor, but excluding microorganisms of the genus Escherichia. The following equation A method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid represented by the formula: 2. The microorganism used is Streptomyces,
The method according to claim 1, which is an actinomycete selected from the genus Streptoverticillium, the genus Mecardia, the genus Nocardiopsis, the genus Saccharopolyspora, the genus Micromonospora, and the genus Streptoporangium. 3. A microorganism used in the genus Bacillus, Micrococcus, Staphylococcus, Pseudomonas,
The method according to claim 1, which is a bacterium selected from the genus Serratia and Corynebacterium. 4. The method according to claim 1, wherein the microorganism used is a yeast selected from the genus Saccharomyces, Candida, Cryptococcus, Debaryomyces, Trigonopsis and Hansenula. 5. A microorganism used in the genus Aspergillus, Mucor, Aureobasidium, Chaetomium,
The method according to claim 1, which is a mold selected from the genus Penicillium and the genus Gliocladium. 6. The microorganism to be used is Streptomyces albus, Streptomyces griseus, Streptomyces hygroscopicus, Streptomyces lividans, Streptomyces viridrochromogenes, Streptomyces pyrosus, Streptoverticillium.・ Cinnamonium, Streptomyces molokaensis,
Nocardia Mediterrani, Nocardiopsis Dassonbelay, Saccharopolispora Hirsta, Micromonospora Carbonacey and Streptosporangium
Actinomycetes selected from Pseudobulgari, or bacteria selected from Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas cepacia, Serratia marcescens and Corynebacterium glutamicum, or Saccharomyces, Seribicii , A yeast selected from Candida albicans, Cryptococcus neoformans, Debaryomyces hanseni, Trigonopsis variabilis, Hansenula schneshi,
Or Aspergillus flavus, Aspergillus
The method according to claim 1, wherein the mold is a mold selected from Terreus, Mucor spinesens, Aurebasidium pullulans, Caetium globosum, Penicillium funiculosum and Gliocladium virens. 7. The microorganism used is Streptomyces hygroscopicus SF-1293 strain (FERM BP-130) or a mutant thereof, Streptomyces hygroscopicus SF-1293 NP-50 strain (FERM BP-1368). ) Or Streptomyces lividans 66 strain (FERM BP-737). 8. The following equation: 4- (hydroxymethylphosphinyl) -2 represented by
Treating oxo-butyric acid with one or more transaminases in the presence of glutamic acid or a salt thereof as amino group donor, characterized by the following formula: A method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid represented by the formula: 9. The enzyme used is 4- (hydroxymethylphosphinyl) -2-oxo in the presence of glutamic acid-oxaloacetic acid-transaminase, glutamic acid-pyruvic acid-transaminase, or glutamic acid as an amino group donor. -Butyric acid is L-2-amino-4
9. The method according to claim 8, which is any one of transaminases converting to-(hydroxymethylphosphinyl) -butyric acid, or a combination of two or more thereof.